Gel electrophoresis is an analytical or preparative technique in which molecules are placed in a gel and an electric field is applied. The molecules are typically charged, and move through the gel under the influence of the electric field. In one embodiment, the gel serves only as an anticonvective matrix, and the molecules separate according to their ratio of charge to mass. More typically, the molecules interact with the gel to a significant extent, and larger molecules experience greater steric hindrance due to encountering the gel, and thus move more slowly. In either case, mixtures composed of multiple species of molecule can be separated into the component species, for either analytical or preparative purposes. When the electric field is turned off, the molecules of a given class are found in discrete areas of the gel ("bands" or "spots"), and can be detected and/or extracted as desired.
Gels for electrophoresis made from in-situ polymerized and cross-linked acrylamide are critical materials in life science research and biotechnology. Protein molecular weights are normally evaluated by electrophoresis on polyacrylamide gels in the presence of denaturing detergents, such as "SDS" (sodium dodecyl sulfate), widely cited in scientific articles. Polyacrylamide gels are also used in the separation of small fragments of DNA. In particular, polyacrylamide gels containing urea or other denaturing agents are used for sequencing of DNA, and are one of the technologies which are critical for the human genome sequencing project.
Current widely-used recipes for making polyacrylamide gels consist of the following steps:
1) Make a solution containing an appropriate concentration of an ethylenically-unsaturated polymerizable water-soluble monomer--most commonly, acrylamide, although other such monomers are used in some systems--and also containing a cross-linking agent (most commonly methylene-bisacrylamide), and appropriate buffers and optional denaturing agents (such as SDS, urea) for the particular type of gel desired.
2) Prepare the gel cassette for casting. Because the polymerization reaction of the prior art is sensitive to oxygen, the solution to be polymerized must be isolated from the atmosphere during polymerization. In addition, the solution is commonly degassed before polymerization is initiated. Because plastic tends to absorb oxygen, which can be released to the solution during polymerization and cause inhibition of polymerization at the plastic surface, the cassettes for casting polyacrylamide gels are normally made of glass, which is heavy and breakable.
3) Add chemical initiators of polymerization to the gel-forming mixture. In current practice these are usually ammonium persulfate, as a source of free radicals, and TEMED tetramethylethylenediamine), an aliphatic tertiary amine which may function as a carrier of radicals from the peroxygen to the double bonds of the the acrylamide molecules.
4) Quickly pour the activated mixture into the cassette. A small amount of surface area of the cassette, usually one surface (typically the top edge of a slab) or two surfaces (the ends of a capillary) are left open to allow access for the activated mixture, which has begun to polymerize. Well-forming combs are often inserted at this stage. The open surfaces may be overlaid with buffer or other solution, both to achieve flatness and to retard diffusion of oxygen into the surface layer of polymerizing mixture.
5) Wait, typically from 30 minutes to several hours, for the polymerization reaction to be completed. During this prolonged polymerization phase, the fluid pre-polymer mixture may leak from the cassette, thereby requiring repetition of the gel casting operation. The reaction speed can be increased by increasing the concentrations of peroxygen and carrier, but then the working time to cast the polymerizing mixture is significantly reduced.
In the early 1960s, polyacrylamide gels were also polymerized by light ("photopolymerized"), using riboflavin or its more soluble derivative, riboflavin phosphate. However, these gels also required hours to polymerize, were also oxygen-sensitive, and the polymerization reaction was no more reliable than the chemically-polymerized system. Riboflavin-catalyzed systems have fallen into disuse, and citations of riboflavin-polymerized gels in the scientific literature are now only historical.
New photopolymerization systems have since been invented, typically for use in the formation of industrial coatings. The photoinitiator 2,2-dimethoxy-2phenyl-acetophenone (DMPAP) and related compounds were disclosed by Sandner and Osborn in U.S. Pat. Nos. 3,715,293 and 3,801,329. These patents disclose acetophenones di or tri-substituted at the 2 position, as improvements over acetophenones substituted at the 3, 4 and/or 440 position, analogous xanthophenones, and benzoin and its lower alkyl derivatives. Also disclosed are coatings, including colored coatings, in which a liquid ethylenically-unsaturated material is polymerized to a hard coating using the 2-acetophenones. Osborn and Tercker, U.S. Pat. No. 3,759,807, disclose the combination of phenones, including certain acetophenones, xanthones, fluoroenones, and anthroquinones, in combination with certain amines, for example triethanolamine, for rapid photopolymerization of unsaturated compounds, including acrylamide. Compositions containing no water are of particular interest in all these applications.
The acetophenones operate by a different mechanism than the flavins. DMPAP and relatives, on excitation by an appropriate wavelength of light, photo-dissociate into a pair of radicals. These appear to be highly effective in the polymerization of unsaturated materials. A recent example of their use is given by Hubbell et al, U.S. Pat. No. 5,410,016, in the synthesis of hydrogels for in-vivo medical applications. Other systems disclosed by Hubbell et. al. as suitable as initiators for photopolymerization in acrylate-derivatized polyethylene glycols include eosin or erythrosin, both derivatives of fluorescein, combined with an amine as transfer agent or co-initiator. The Hubbell and the Osborn patents are hereby incorporated by reference.
Berner and Manse (U.S. Pat. No. 4,609,612) disclose supplementation of benzophenones with benzoylcyclohexanol in photopolymerization. Reilly (U.S. Pat. No. 4,576,975) discloses carboxylated analogs of "Mitchler's ketone", a diaminobenzophenone, as watersoluble photoinitiators. Moffat et al (U.S. Pat. No. 5.449,724) and Li et al (Macromolecules 28:6692-93, 1995) disclose the use of nitroxides as initiators, although not as photoinitiators.
Various monomers can be used in addition to the conventional acrylamide/bis-acrylamide solution. It is known in conventional chemically-polymerized gels to use hydroxyethylmethacrylate and other low-molecular weight acrylate-type compounds as monomers; these have been commercialized as "Lone-Ranger" gels. Use of polymers substituted with one or more acrylate-type groups has also been described in the literature (Zewert and Harrington, Electrophoresis 13:824-831, 1992), as especially suitable for separations in mixed solvents of water with miscible organic solvents, such as alcohol or acetone.
In DNA electrophoresis, the most commonly-used gels are made of agarose, a seaweed extract. This material forms a thermoreversible gel on reduction of temperature. Since the reaction is not affected by oxygen, gel cassette design is less constrained, and gels are often cast in plastic containers, and with the upper face exposed to air or buffer ("submarine gels"). However, agarose does not generally have as fine a resolving capacity as acrylamide and many gels for separating nucleic acids, such as DNA sequencing gels, are made by peroxygen-catalyzed polymerization of acrylamide. Since conventionally polymerized acrylamide gels (using peroxygens or riboflavins) cannot be cast in the presence of significant oxygen, the options available for casting techniques are limited. In particular, the "open-face" gels typical of agarose, with 30 to 50% of their surface exposed to oxygen during polymerization (and the rest of the surface area often exposed to plastic), cannot currently be made with ethylenically-unsaturated monomers.
There are other considerations which must be satisfied in casting a gel suitable for the electrophoretic separation of molecules.
First, the gel must have a suitable "format", in that it must be of an appropriate geometrical shape to perform the separation, and it must be placed in a suitable container, or electrophoresis cell, to allow the electric field to be applied, and for excess heat and evolved gas to be removed from the separation area. Gels for electrophoresis are generally either flattened rectangles, cast in a rectangular open box or closed cassette, or are cast as cylinders inside a tube. In current practice, tubes are generally of capillary dimensions (20-200 .mu.meter (.mu.) in inner diameter).
Second, the gel must be generally uniform. Large fluctuations in the local concentration of the gel are clearly undesirable, and can significantly distort a separation (except where deliberately introduced, as in a "step" gradient gel.) A particularly troublesome microvariation is the formation of bubbles during the polymerization; these can sharply degrade performance, especially in capillaries. Local microvariations also degrade performance by causing excessive band broadening during the separation. Such microvariations are thought to arise from heating of the solution during polymerization. Conventional peroxygen polymerization reagents liberate additional heat during the polymerization reaction, further increasing the tendency towards distortions which are inherent in the normally exothermic polymerization of ethylenically unsaturated molecules.